INTRODUCTION
Point mutations are the most common events in human genetic diseases and nearly 50% of disease-associated mutations are C>T and G>A substitutions (Gaudelli et al., 2017). Animal modeling of human genetic diseases are valuable in study of pathogenic mechanism and testing of drug efficacy. CRISPR Cas9 system is an adaptive immune system in bacteria that protects its genome from invading viruses (Rath, Amlinger, Rath, & Lundgren, 2015; Sontheimer & Barrangou, 2015). CRISPR Cas9 system has been successfully applied to genetic engineering in other cells and organisms. It is as well utilized to precisely correct or introduce point mutations via homology-directed repair (HDR). It requires DNA double-strand breaks (DSBs) at the target and a DNA template with homologous arms (Wu et al., 2018; Y. Yang et al., 2016; L. Yang et al., 2013; Yin et al., 2014). However, cells respond to DSBs more often with nonhomologous end joining (NHEJ) that may introduce insertions or deletions (indels) and lead to disruption of corresponding genes (Davis & Chen, 2013; Komor et al., 2017).
CRISPR/Cas9-based cytidine base editors (CBEs) have recently been developed to generate precise base changes with high efficiency (Gaudelli et al., 2017; Komor, Kim, Packer, Zuris, & Liu, 2016; Nishida et al., 2016; Ma et al., 2016). CBEs system consists of a CRISPR-Cas9-derived DNA-binding module and a cytidine deaminase, and is able to introduce nucleotide substitutions of C>T (Xie et al., 2019; K. Kim et al., 2017; D. Kim et al., 2017) and G>A (Zhen Liu et al., 2018) at targeted loci without need of DSBs. It has been demonstrated successfully in various organisms (D. Kim et al., 2017).
Base editing systems have gone through various stage of improvement to broaden their applicability and utility in editing of single nucleotide and have been widely applied to cell lines, various animals and plants. The fourth generation of base editor 4 (BE4) has a cytidine deaminase (rAPOBEC1) with two copies of uracil glycosylase inhibitor (UGI) that are directly fused to C terminus of Cas9n, a Cas9 mutant with a D10A amino acid substitution, through a 32 amino acid linker (Fig. 1A). BE4 enables direct conversion of cytidine (C) to uridine (U) in chosen bases of DNA sequence (Komor et al., 2017). However, feasibility and efficacy of this system has not been assessed in mice. In the current study, we have confirmed that BE4 system is able to perform a multiplexed base editing with high precision and efficiency in mice. BE4 system shows great potentials in modeling human genetic diseases.